Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into the three germ layers and possibly all tissues of the human body (Strelchenko et al., 2004, Reprod Biomed Online 9: 623-9; D'Amour et al., 2000, Nat Biotechnol 18: 381-2; Keller et al., 1999, Nat Med 5: 151-2; Trounson, 2002, Reprod Biomed Online 4 Suppl 1: 58-63; Odorico et al., 2001, Stem Cells 19: 193-204; Gertow et al., 2004, Stem Cells Dev 13: 421-35). hESCs were originally isolated from the inner cell mass of human embryos and were found capable of passaging through over 100 divisions while maintaining pluripotency (Thomson et al., 1998, Curr Top Dev Biol 38: 133-65; Reubinoff et al., 2000, Nat Biotechnol 18: 399-404). In addition to protocols for culturing hESCs in an undifferentiated state, differentiation protocols for hESCs have been successfully established in vitro for many cell types, including neuronal cells (Park et al., 2005, J Neurochem 92: 1265-76), hematopoietic cells (Kaufman et al., 2002, J Anat 200: 243-8), insulin-producing cells (Assady et al., 2001, Diabetes 50: 1691-7), endothelial cells (Levenberg et al., 2002, Proc Natl Acad Sci USA 99: 4391-6), and cardiomyocytes (Mummery et al., 2002, J Anat 200: 233-42), among others (Odorico et al., 2001, Stem Cells 19: 193-204; Reubinoff et al., 2000, Nat Biotechnol 18: 399-404).
The ability of hESCs to differentiate into many cell types distinguishes them from adult stem cells, which can typically only differentiate into limited cell types (Thomson et al., 1998, Curr Top Dev Biol 38: 133-65; Reubinoff et al., 2000, Nat Biotechnol 18: 399-404). Thus, hESCs provide a particularly useful system for studies of development. hESCs also have enormous therapeutic potential for treatment of a wide variety of diseases, including Parkinson's disease, diabetes and heart failure, among others
To make hESCs compatible for clinical therapy, banks of hESC lines with different human leukocyte antigens (HLA) are being established to enable HLA matches to reduce the likelihood of graft rejection by a transplant recipient (ISSCR, 2002, The ISSCR Newsletter-The Pulse Vol 1(3) Nov. 1, 2002; National Institutes of Health, 2005, “NIH Awards a National Stem Cell Bank and New Centers of Excellence in Translational Human Stem Cell Research” Oct. 3, 2005 Press Release; Taylor et al., 2005, Lancet 366: 2019-25). In addition, other technologies, such as nuclear transfer, may allow the generation of autologous embryonic stem cells in the future (Wakayama et al., 2001, Science 292: 740-3). Thus, hESCs are expected to provide a great resource for regenerative medicine (Dvash et al., 2004, Best Pract Res Clin Obstet Gynaecol 18: 929-40).
Until recently, hESC lines were derived and proliferated in culture medium containing animal products. The presence of xenograft or allograft animal products in hESC culture media has at least four problems (Pera, 2005, Nat. Methods 2:164-165; Draper et al., 2004, Stem Cells Dev. 13:325-336; Stojkovi et al., 2004, Reproduction 128:259-267). First, animal products may contain toxic proteins or immunogens that evoke an immune response in the recipient and thus lead to rejection upon transplantation (Martin et al., 2005, Nat Med 11: 228-32). Second, the use of animal products increases the risk of contamination by animal pathogens, such as viruses or prions, which could endanger the recipient (Cobo et al., 2005, Appl Microbiol Biotechnol 68: 456-66). Third, separating animal products, such as feeder cells, from hESCs is both time- and labor-intensive. Finally, the use of medium with undefined factors greatly complicates developmental studies, for instance, by undermining the predictability of culture conditions and possibly leading to undesirable cell differentiation. Therefore, there is a great need for a defined medium with animal products that supports growth of hESCs without substantial differentiation, and while maintaining pluripotency.
To date, four key components required for hESC culture have been identified. First, basic fibroblast growth factor (bFGF) has been shown to be essential for hESC self-renewal (Granerus et al., 1996, Cell Prolif 29: 309-14; Xu et al., 2005, Stem Cells 23: 315-23). Second, feeder cells, conditioned medium, or cytokines, such as transforming growth factor (TGF) (Beattie et al., 2005, Stem Cells 23: 489-95) or Wnt3 (Sato et al., 2004, Nat Med 10: 55-63), are necessary. Third, an extracellular matrix is necessary. Fourth, fetal bovine serum or serum replacement (Holden, 2005, Science 307: 1393; Xu et al., 2001, Nat Biotechnol 19: 971-4) is necessary.
Several types of matrices have been used to coat the culture dish surface for hESC culture. BD Matrigel™ (BD Biosciences, San Jose, Calif.), a preparation rich in multiple extracellular components, is secreted by mouse Engelbreth Holm-Swarm sarcoma cells and is able to support hESC growth (Xu et al., 2001, Nat Biotechnol 19: 971-4). Matrigel™ contains laminin, collagen type IV, heparan sulfate, proteoglycan, and entactin (Kleinman et al., 1986, Biochemistry 25: 312-8). Human serum can substitute for Matrigel™, thus avoiding a xeno component in hESC culture (Stojkovic et al., 2005, Stem Cells 23: 895-902). However, both Matrigel™ and human serum are mixtures with undefined components. Other defined matrices, such as fibronectin, laminin, and collagen, can support feeder-cell-free hESC growth, but the efficacy varies among laboratories, and some reagents have disparities among different lots (Xu et al., 2001, Nat. Biotechnol. 19: 971-4; Ludwig et al., 2006, Nat. Biotechnol. 24: 185-187; Li et al., 2005, Biotechnol. Bioeng. 91: 688-98).
Serum or serum replacement is also essential for hESC culture. Knockout™ SR (Invitrogen, Carlsbad, Calif.), which contains animal-derived products, is a serum replacement frequently used in hESC culture (Xu et al., 2005, Stem Cells 23: 315-23). An animal-free product, X-VIVO™ (Cambrex Bio Science, Walkersville, Md.) supports hESC growth, however it was optimized for hematopoietic cell culture (Li et al., 2005, Biotechnol Bioeng 91: 688-98). Disadvantageously, Knockout™ serum and X-VIVO™ are both proprietary materials and contain multiple components. Moreover, in feeder-cell-free culture, hESCs grown in medium containing these serum replacements form differentiated cells around the hESC colonies, indicating that optimal conditions have not been achieved (Li et al., 2005, Biotechnol Bioeng 91: 688-98).
Because of these problems with currently known culture media for hESC, there is a need for a better, defined culture medium with minimal components that reproducibly supports robust growth of hESCs. The present invention meets these needs.